The effect of actinomycin D reflects simply a blockage of RNA synthesis. The effect of cycloheximide may reflect the requirement for a newly synthesized protein factor in the signaling pathway for induction of the gene encoding this mRNA.

The N-terminal amino acid residues of Sxl expressed from P_e (encoded by exon E1) differ from the N-terminal residues of Sxl expressed from P_m (encoded by exon L2).

The RNA-binding site for proteins that carry out 3′-end cleavage and polyadenylation is defined most reliably by the sequence AAUAAA. In the DNA, the sequence is AATAAA, located downstream from the final gene exon. Because AATAAA is a consensus sequence, scanning only for this sequence would not find all the 3′-end cleavage sites; some will vary slightly from the consensus.

Reticulocytes are the precursors of red blood cells, filled with hemoglobin and highly specialized for oxygen transport; their nucleus is destroyed during maturation. Almost all the mRNA deposited in a reticulocyte before destruction of the nucleus encodes hemoglobin. There are essentially no other translation-dependent cellular functions to disrupt.

S-22

(a) AAUAAA is a signal for 3′-end cleavage and polyadenylation. (b) AUUUA is an ARE motif that limits mRNA stability.

Sequences called intronic splicing silencers, or ISSs, are bound by proteins that suppress splicing at these sites.

(1) mRNAs could be deposited at one end of the oocyte, such as by Drosophila nurse cells. (2) Proteins could be deposited at one end of the oocyte. (3) mRNAs or proteins could be actively transported from one part of the oocyte to another. (4) A set of mRNAs or proteins could be subjected to differential stability by the introduction, at one end of the oocyte, of factors leading to mRNA or protein degradation.

Anterior cells of the embryo, where Mex-3 is concentrated and normally suppresses Pal-1 production, would take on fates similar to those of cells at the posterior end.

The bcd mRNA needed for development is contributed to the egg by the mother. The fertilized egg develops normally, even if its genotype is bcd⁻/bcd⁻, as long as the mother has one wild-type bcd allele (and thus contributed the mRNA to the oocyte) and the bcd⁻ allele is recessive. However, an adult bcd⁻/bcd⁻ female will be sterile, because she cannot generate bcd mRNA for her oocytes.

The cap cells may create a niche for the germ-line stem cell, providing extrinsic signals that orient the cell division to ensure that one daughter retains the stem cell identity. In this case, the cap cells supply protein ligands that activate a Bmp-family signaling pathway in the stem cell that represses at least one gene required for differentiation.

An embryonic stem cell is a cell, derived from a mammalian embryo, that has the potential to differentiate into almost any kind of tissue. An induced pluripotent stem (iPS) cell is a somatic cell, such as a skin cell, that has been reprogrammed to have pluripotent potential to differentiate into different tissue types.

The ability to feed bacteria to the worms allows an RNAi approach. Given knowledge of the genome sequence and the types of genes that might be involved (e.g., Wnt-class signaling genes, homeotic genes), you could devise an RNAi screen. Short double-stranded RNAs complementary to worm genes are expressed in bacteria, using gene segments cloned between opposing promoters on a bacterial plasmid, and the bacteria are fed to worms. The heads of the fed worms are cut off, and the RNAi clones that affect regeneration are determined. Such an experiment has been done, with the finding that one homeobox gene of the piwi family plays a key role. (See P. W. Reddien et al., Dev. Cell 8:635–649, 2005; P. W. Reddien et al., Science 310:1327–1330, 2005.)

(a) As described in Chapter 7, the three-hybrid method is designed to screen for RNA sequences that bind to a particular protein. The method used here was modified to screen for unknown proteins that bind to a particular RNA sequence. (b) The second control from the top shows that a single nucleotide change in UCUUU abolishes binding. (c) FBF-1 may be binding to some other sequence in the engineered RNA, and the various control sequences help eliminate that possibility. (d) The FBF-1 not only localizes to the germ line but is predominantly present in the cytoplasm. (e) The animals should (and most do) produce only sperm.